The cytotoxic T lymphocyte (CTL) response is a major component of the immune system, active in immune surveillance and destruction of infected or malignant cells and invading organisms expressing foreign antigens on their surface. The ligand of the antigen specific T cell receptor is a complex made up of a peptide fragment of an antigen bound to major histocompatibility complex (MHC) molecules. In particular, cytotoxic T lymphocytes recognise peptide bound to MHC Class I molecules.
MHC class I molecules are normally expressed at the cell surface as ternary complexes formed by a heavy chain of 46 kD, a light chain called β2-microglobulin (β2M) of 12 Kd and a peptide composed of 8-10 amino-acids Ivan Bleek, G. M. and S. G. Nathenson, Nature 348:213, 1990; Zhang, W. et al., Proc. Natl. Acad. Sci. USA 89:8403, 1992; Matsumura, M. et al., Science 257:927, 1992; and Latron. F., et al., Science 257:964, 1992). Formation of the ternary complex is thought to involve transport into the lumen of the endoplasmic reticulum (ER) of peptides generated by protein degradation in the cytoplasm (Nuchtern. J. G. et al., Nature 339:223, 1989; Yewdell, J. W. and J. R. Bennink, Science 244:1072, 1989; and Cox. J. H. et al., Science 247:715, 1990). The study of mutant cell lines selected for their low expression of MHC class I molecules at the cell surface has provided insights into the molecular events required for antigen processing. These studies have allowed the identification of two genes located in the MHC region which encode proteins of the ATP binding cassette (ABC) family. These genes, called TAP-1 and TAP-2, have been implicated in transport of peptides from the cytoplasm to the lumen of the ER (Deverson, E. V. et a). Nature 348:738, 1990; Trowsdale, J. et al., Nature 348:741, 1990; Spies, T. et al., Nature 348:744, 1990; Monaco, J. J. et al., Science 250:1723, 1990; Spies, T. and R. DeMars, Nature 351:323, 1991; Bahram, S. et al., Proc. Natl. Acad. Sci. USA 88:10094, 1991; Spies, T. et al., Nature 355:644, 1992; Kelly, A. et al., Nature 355:641, 1992; Powis, S. H. et al., Proc. Natl. Acad. Sci. USA 89:1463, 1992; and Colonna. M. et al., Proc. Natl. Acad. Sci. USA 89:3932, 1992). Two other MHC linked genes. LMP-2 and -7 (Monaco, J. J. and McDevitt, 1982, Proc. Natl. Acad. Sci. USA 79:3001), are components of the proteasome, a cytoplasmic multicatalytic protease complex, which is likely responsible for some aspects of protein degradation for antigen processing (Ortiz-Navarette, V. et al., Nature 353:662, 1991; Brown, M. G. et al., Nature 353:355, 1991; Glynne, R. et al., Nature 353:357, 1991; Martinez, C. K. and J. J. Monaco, Nature 353:664, 1991; Kelly, A. et al., Nature 353:667, 1991; Yang, Y., et al., Proc. Natl. Acad. Sci. USA 89:4928, 1992; Goldberg, A. L. and K. L. Rock, Nature 357:3751 1992).
The mouse mutant lymphoma cell line RMA-S expresses low levels of class I molecules at the cell surface compared to the wild type RMA cells (Ljunggren, H.-G. et al., J. Immunol. 142:2911, 1989; and Townsend. A et al., Nature 340:443, 1989). Influenza virus infected RMA-S cells present influenza peptides in the context of Db molecules inefficiently and are only weakly recognized by specific CTL (Townsend, A et al., Nature 340:443, 1989). Transfection with the putative transporter gene, TAP-2, complements this deficiency (Powis. S. J. et al., Nature 354:528, 1991; and Attaya. M. et al., Nature 355:647, 1992). The endogenous TAP-2 gene of RMA-S cells was shown to contain a point mutation which introduces a stop translation codon resulting in an incomplete and defective TAP-2 protein (Yang. Y. et al., J. Biol. Chem. 267:11669, 1992). Despite the defective TAP-2 protein in RMA-S cells, antigenic peptides from vesicular stomatitis virus (VSV) bypass the defect and are presented to specific CTL by Kb molecules in RMA-S cells (Esquivel. F., et al., J. Exp. Ned. 175:163, 1992; and Hosken. N. A. and M. J. Bevan, J. Exp. Med. 175:719, 1992). The VSV-nucleocapsid (N) peptide, VSV-N 52-59, has been shown to be the major peptide presented by Kb molecules on VSV infected cells (van Bleek, G. M. and S. G. Nathenson, Nature 348:213, 1990). The presence of the wild-type TAP-1 protein in RMA-S cells may be sufficient for translocation of the VSV-N 52-59 peptide to the ER lumen (Powis. S. J. et al., Nature 354:528, 1991; Attaya, M. et al., Nature 355:647, 1992; and Yang. Y. et al., J. Biol. Chem. 267:11669, 1992). Alternatively, the VSV-N 52-59 peptide may not need a functional transporter for transport into the lumen of the ER. Expression of minigene-encoded viral peptide epitopes in T2 cells (Zweerink. H. J. et al., J. Immunol. 150:1763, 1993) and in-vitro translation and translocation using microsomes from T2 cells (Lévy, F. et al., Cell 67:265, 1991) support this contention.
A separate class of antigen processing variants are those in which the assembly and the surface expression of MHC class I molecules are entirely inducible by IFN-γ (Klar, D. and G. J. Hc1 immering, EMBO J. 8:475, 1989). For example in the small lung carcinoma cell line. CMT.64, recognition by influenza virus specific CTL does not take place unless induced with IFN-γ (Sibille, C. et al., Eur. J. Immunol. 22:433, 1992). The very low amount of all proteasome components present in uninduced CMT.64 cells is presumed to be responsible for their phenotype (Ortiz-Navarette. V et al., Nature 353:662, 1991). Exogenous influenza peptides can bind to Db molecules on CMT.64 cells and complement recognition by influenza specific CTL (Sibille, C. et al., Eur. J. Immunol. 22:433, 1992). In addition, it has been found that the β2m and the VSV-N 52-59 peptides added exogenously to these cells complement recognition by VSV specific CTL restricted to Kb (Jefferies W. A et al., 1993, J. Immunol. 151:2974). The amount of β2m and of heavy chains synthesized in these cells may limit the amount of MHC class I expression on the cell surface (Jefferies et al, supra, 1993). A dysfunction of the putative peptide transporters and/or in the generation of the peptide may be responsible for the CMT.64 phenotype which may represent a mechanism to downregulate MHC class I expression, a feature common to many carcinomas.
Restifo, N. R. et al. (J. Exp. Ned. 177:265-272, 1993) studied the antigen processing efficiency of 26 different human tumor lines using a recombinant vaccinia virus (VV) to transiently express the Kd molecule. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to Kd-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by the cell lines from the endoplasmic reticulum (ER) to the cell surface. Northern blot analysis of the cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP7 and LMP-2 as well as the putative peptide transporters TAP-1 and TAP-2.
There is a need in the art for methods to augment or enhance an immune response to various targets including virally infected and malignant cells.